The introduction of genetic material into living cells is a common natural occurrence. A wide variety of biological mechanisms have evolved to facilitate, regulate, or prevent the incorporation of new DNA sequences by living cells. Changes in cellular phenotype which result from such events may profoundly influence the characteristics of the cell, including its ability to live, to cause disease, and to produce beneficial or harmful products.
As a consequence, a large amount of effort has been expended in the last 40 years to develop methods by which exogenous genetic information (hereafter referred to as "exogenous DNA", although in some cases the genetic information may be encoded in RNA) can be introduced at will into cells in such a way as to effect desired changes in cellular properties of an organism. These processes of introduction of exogenous DNA into cells are referred to as transformation, transfection, convection, etc. (reviewed in Smith, Danner and Deich, Ann. Rev. Biochem., 50, 41 (1981)). Some of these methods are based on naturally occurring mechanisms of DNA uptake which occur in certain types of bacteria (Avery, McLeod, and McCarty, J. Exp. Med., 79, 137 (1944)), are based on the use of viruses which can infect susoeptible host cells (Zinder and Lederberg, J. Bacteriology, 64, 679 (1952)), or are based on bacterial strains of A. tumafaciens which can infect and transfer plasmid DNA into the cells of a susceptible plant species (Chilton, et al., Cell, 11, 263 (1977)). Other methods for introducing DNA into cells have also been described based on treatment of cells with calcium chloride (Mandel and Higa, J. Mol. Biol., 53, 159 (1970)), the use of polyethylene glycol (PEG) (Chang and Cohen, Mol. Gen. Genet., 168, 111 (1979)), in vitro packaging of DNA in viral coats (Fraenkel-Conrat and Williams, Proc. Natl. Acad. Sci. USA, 41, 690 (1955); Hohn and Murray, Proc. Natl. Acad. Sci. USA, 74, 3259 (1977)), fusion with cells of DNA-containing liposomes (Fraley et al., Proc. Natl. Acad. Sci. USA, 76, 3348 (1979)) or protoplasts (Schaffner, Proc. Acad. Natl. Acad. Sci. USA, 77, 2163 (1980)) or by mechanical introduction of DNA by microinjection (Capecchi, Cell, 22, 479 (1980)).
In the case of single-celled organisms or the isolated cells of multi-cellular organisms grown in culture, reliable methods for the introduction of exogenous DNA have been described in the literature and are widely used. These include the calcium chloride-mediated uptake of DNA in E. coli (Mandel and Higa, J. Mol. Biol., 53, 159 (1970)), polyethylene glycol-mediated uptake in yeast (Hinnen et al., Proc. Natl. Acad. Sci. USA, 75, 1929 (1978) and CaPO.sub.4 -mediated uptake in cultured cells (Graham and van der Eb, Virology, 52, 456 (1973); Pellicer et al. Science, 209, 1414 (1980)). These methods generally result in the successful introduction of exogenous DNA into a very small fraction of the cells exposed to the exogenous DNA, generally on the order of 1 cell in 10.sup.4 or less. These methods are therefore used in combination with a methodology for selecting cells into which the exogenous DNA has become incorporated and thereby the cells containing the exogenous DNA have acquired a distinctive property, such as resistance to a particular antibiotic or the ability to utilize a particular growth medium, not possessed by a similar cell which has not incorporated the exogenous DNA. Since even small cultures of bacterial or cultured cells can easily contain 10.sup.5 or more cells, this approach readily yields at least a few cells which have taken up the exogenous DNA and which can be grown to increase the number of cells containing the exogenous DNA.
It is impractical to apply a similar approach to that which has been widely used for bacteria to the transformation of the cells of multi-cellular animals. The cells of multi-cellular organisms are differentiated and even if the cells in the various intact tissues could be somehow rendered permeable to exogenous DNA uptake, the low frequency of success would be unlikely to result in the acquisition by the organism of any useful characteristics. A more promising approach would be to attempt to transform cells of the germ line (in animals) or of cells (from a plant) which could be regenerated into a complete multi-cellular organism. Germ line cells can not usually be grown in culture, nor can large numbers be easily tested for the presence of exogenous DNA. Consequently, a method of transfer whose efficiency is inherently higher than that of methods widely used with free-living single cells is highly desired.
One approach which might be considered is to use the standard methods of transformation on a cell population. The desired transformed cells are then selected and grown. Subsequently, the transformed cells can be used to generate a complete organism carrying the incorporated DNA. This approach has been widely discussed in the case of plant protoplasts from certain plant species which can be used to regenerate complete whole plants (Steward, Proc. Royal. Soc. Lond. (Biol.), 175, 1 (1970); Vasil and Hildebrant, Science, 150, 889 (1975)). The component steps of this process described immediately above have been shown to be feasible in the case of certain lines of mouse teratocarcinoma cells. The uptake and function of DNA in one line of cultured teratocarcinoma cells has been demonstrated (Pellicer et al. Proc. Natl. Acad. Sci. USA, 77, 2098 (1980)), and cells of a different line have been used to generate fertile adult mice (Stewart and Mintz, Proc. Natl. Acad. Sci. USA, 78, 6314 (1981)). Adult mice containing transformed DNA have yet to be produced in this manner, however.
An alternative approach which has been suggested is to transform cells such as lymphocytes which can repopulate a portion of an adult organism, i.e., bone marrow (Cline et al., Nature (Lond.), 284, 422 (1980); Mercola et al. Science, 208, 1033 (1980)). The presence of transferred genes in the repopulated bone marrow cells has not yet been demonstrated, however.
Only a very limited number of organisms can be regenerated from cells which can be propagated in vitro in culture. Thus, a far more generally applicable approach would be to directly introduce exogenous DNA into an egg or early embryo of a multi-cellular organism. After development has been completed, the tissues of the resulting organism are then evaluated for the presence of the incorporated exogenous DNA. If germ line cells are among those cells in which exogenous DNA has been retained, some of the progeny may contain the exogenous DNA in all of their cells.
The approach described immediately above requires methods for the introduction of exogenous DNA which are significantly more efficient than those methods used with populations of single cells in culture. In principle, any of a wide variety (see Hinnen et al., (1977); Pellicer et al., (1980); Capecchi, (1980); Fraley et al., (1979); Chang and Cohen, (1979); Hohn and Murray (1977); supra) of presently available techniques could be used to introduce exogenous DNA into eggs, zygotes, or other germ line cells. Certain viruses infect early embryonic cells, including those of the germ line, and viral genetic information may become integrated into the cellular chromosomes. This gives rise to organisms which stably transmit this originally exogenous DNA to their progeny and may display characteristics (i.e., virus production) associated with the originally exogenous DNA (Jaenisch, Proc. Natl. Acad. Sci. USA, 73, 1260 (1976); Harbers et al., Nature (Lond.), 23, 540 (1981)). Specific insertion of DNA by this approach has so far only been used to transfer viral genetic information.
One of the potentially most efficient means of gene transfer into a multi-cellular organism is direct microinjection of exogenous DNA into cell nuclei (Capecchi, Cell, 22, 479 (1980)). Several investigations have recently described the retention of exogenous DNA in adult mice which developed from fertilized eggs that had received DNA injected into one of the pronuclei (Gordon et al., Proc. Natl. Acad. Sci. USA, 77, 7380-84 (1980); Wagner, Stewart and Mintz, Proc. Natl. Acad. Sci. USA, 78, 5016 (1981); Brinster et al., Cell, 27, 223 (1981); Constantini and Lacy, Nature (Lond.), 294, 92 (1981); Wagner et al., Proc. Natl. Acad. Sci. USA, 78, 6376 (1981)). In some of these mice the transferred exogenous DNA was transmitted to its progeny and showed signs of activity. Retention of exogenous DNA during development following injection into fertilized frog eggs has also been reported (Rusconi and Schaffner, Proc. Natl. Acad. Sci. USA, 78, 5051 (1981)).
The currently available conventional methods for introducing exogenous DNA into eukaryotic cells and organisms as described above suffer from numerous difficulties which limit their use. Some of the important limitations are set forth below:
(1) It is not possible in any of these methods to precisely control and to vary at will what exogenous DNA sequences are transferred into the genome of the recipient;
(2) Those exogenous DNA sequences which are transferred into the genome of the recipient have often undergone changes in structure and/or number during the process of transfer or during subsequent propagation of the recipient;
(3) The transfer of DNA sequences into the genome of a recipient is not known to depend on the presence of catalytic factors which are not present in the recipient cell itself, and, thus, it is not likely to be possible to limit transfer of DNA sequences to specific tissues or developmental stages of the recipient;
(4) Transfer of DNA sequences into the genome of the recipient occurs at low frequencies in most of the conventional methods; and
(5) Genes introduced into multicellular organisms by conventional methods have never been shown to be subject to normal developmental controls, however, this is likely to be a prerequisite for most applications in such multicellular organisms.
Accordingly, development of a system which overcomes or significantly reduces the above difficulties of conventional methods of DNA incorporation based on a unique utilization of the properties of natural biological entities known as transposons or transposable elements (reviewed in Calos and Miller, Cell, 20, 579 (1980)) is desired.